COPYRIGHTpsasir.upm.edu.my/id/eprint/76338/1/FPV 2018 24 IR.pdf · 2019. 12. 2. · meningkatkan...

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UNIVERSITI PUTRA MALAYSIA

COCKLE SHELL-DERIVED NANO CARRIER FOR ARA-C IN THE TREATMENT OF ACUTE MYELOID LEUKAEMIA

MUSTAFA SADDAM GHAJI

FPV 2018 24

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PMCOCKLE SHELL-DERIVED NANO CARRIER FOR ARA-C IN THE

TREATMENT OF ACUTE MYELOID LEUKAEMIA

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of

Doctor of Philosophy

June 2018

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos, icons,

photographs, and all other artwork, is copyright material of Universiti Putra Malaysia

unless otherwise stated. Use may be made of any material contained within the thesis

for non-commercial purposes from the copyright holder. Commercial use of material

may only be made with the express, prior, written permission of Universiti Putra

Malaysia.

Copyright© Universiti Putra Malaysia

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DEDICATIONS

To

My Dear Parents,

Mr. Saddam Ghaji

Mrs. Sbeha Abod

To

My brothers and sisters

To

My Marvellous Family

In Recognition of their Worth, Love, and Respect

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfilment

of the requirements for the degree of Doctor of Philosophy

COCKLE SHELL-DERIVED NANO CARRIER FOR ARA-C IN THE

TREATMENT OF ACUTE MYELOID LEUKAEMIA

By


June 2018

Chairman : Professor Md Zuki bin Abu Bakar, PhD

Faculty : Veterinary Medicine

Leukemia is a cancerous disease of bone marrow and blood in which acute form

progresses more rapidly than the chronic form. The major therapeutic approaches of

different cancer types are limited to conventional chemotherapy such as (Ara-C) which

suffers less specific, high toxicity and short half-life, multidrug resistance and

selectivity, narrow therapeutic index and significant increases in high dose distribution

to healthy cells or tissues. Targeting anticancer drug delivery system has the potential

to overcome these significant drawbacks by improving chemotherapy drug efficacy,

specific tumor targeting, enhance accumulation in tumor tissues or cells and minimize

the systemic toxicity. Nanoparticles as drug delivery system enable unique approaches

to cancer treatment. Over the last two decades, a large number of nanoparticle delivery

systems have been developed for cancer therapy including organic and inorganic

materials. Cockle shells (Anadara granosa) are found to be a rich natural resource for

calcium carbonate aragonite. In this study, the cockle shell-derived calcium carbonate

aragonite nanoparticles (CCANPs) were used as a carrier for Cytarabine (Ara-C) as a

unique approach for cancer treatment. Nanoparticles were spherical-shaped when

CCANPs was synthesized using the combination of chemical and mechanical method.

The morphology and compositions of the products were characterized by Field

Emission Scanning Electron Microscope (FE-SEM), Transmission Electron

Microscope (TEM), Energy Dispersive X-ray (EDX), X-Ray Diffraction (XRD),

Fourier Transform Infra-Red (FT-IR) and zeta potential. The anti-leukemia drug (Ara-

C) was loaded into CCANPs. The spectrophotometer was used with a wavelength UV-

invisible, to estimate the amount of loading and release profile of Ara-C. The results

showed that the drugs (Ara-C) could be efficiently loaded into the CCANPs, and

furthermore, the fast and sustained release of Ara-C was observed from the

nanocarriers at pH 4.8 and slow release at pH 7.4, which shows pH-dependent

properties. The nanoparticles were used as a carrier against HL-60

human leukemia cells (in vitro study) and for cancer therapy in a murine xenograft

model (SCID mice) (in vivo study). The in vitro evaluation showed IC50 values upon

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72 hours of treatment with pure Ara-C was 5μg/mL, and Ara-C loaded CCANPs was

2.5μg/mL. Apoptosis was demonstrated by Cell Counting Reagent (SF), Flow

Cytometry (FCM), Methylene blue (MB) and Fluorescent Microscope (FM) where

apparently cellular uptake of Ara-C/CCANPs through endocytosis indicating a dose

and time-dependent response relationship. Morphological observations by SEM

revealed microvilli disappearance, cell shrinkage, membrane blebbing and the

formation of apoptotic bodies, which confirmed both Ara-C and half dose of Ara-

C/CCANPs induced apoptosis of HL-60 cells. In brief, Ara-C loaded CCANPs are

more effective than pure Ara-C to human leukemia (HL-60) cells. In vivo study

revealed that CCANPs nanocarrier significantly enhances the effects of Ara-C on

AML through blood smear, bone marrow smear and histopathological survey for vital

organs (heart, liver, lung, spleen and kidney) for severe combined immunodeficient

(SCID) mice. The pharmacokinetic study showing significant effect between pure

Ara-C 50mg/kg group, 100mg/kg CCANPs loaded with 50mg/kg Ara-C and half dose

of loaded drug (25/50 mg/kg), the rate of release of the drug in the plasma was slow

in the two groups of the drug-loaded compared to the pure drug. The study revealed a

new biodegradable, biocompatible, non-toxic to health and pH-sensitive, CCANPs

with a feasible promising potential for targeted delivery carriers of antitumor drugs.

The results established strong evidence that CCANPs has excellent properties that

make it an ideal candidate for biological drug delivery systems.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PEMBAWA NANO BERASASKAN KULIT KERANG UNTUK ARA-C

DALAM RAWATAN LEUKIMIA MYELOID AKUT

Oleh


Jun 2018

Pengerusi : Profesor Md Zuki bin Abu Bakar, PhD

Fakulti : Perubatan Veterinar

Leukemia adalah penyakit kanser sumsum tulang dan darah dimana bentuk akut

merebak lebih pantas dari bentuk kronik. Pendekatan rawatan utama untuk jenis

kanser yang berlainan adalah terhad kepada kemoterapi seperti (Ara-C) konvensional

yang kurang spesifik, tinggi toksisiti dan berjangka hayat yang pendek, bersifat

memilih dan rintangan drug pelbagai, indeks terapeutik yang sempit dan peningkatan

pengedaran dos yang tinggi kepada sel atau tisu yang sihat. Sistem penyampaian drug

antikanser yang disasarkan berpotensi untuk mengatasi permasalahan ini dengan

meningkatkan keberkesanan drug kemoterapi, sasaran tumor yang spesifik,

meningkatkan pengumpulan drug pada tisu atau sel tumor dan mengurangkan toksisiti

sistemik. Nanopartikel sebagai sistem penyampaian drug memungkinkan pendekatan

unik kepada rawatan kanser. Selama dua dekad yang lalu, sebahagian besar sistem

penyampaian nanopartikel untuk terapi kanser telah dicipta termasuk penggunaan

bahan organik dan bukan organik. Kulit kerang (Anadara granosa) merupakan sumber

aragonit kalsium karbonat yang tinggi. Di dalam kajian ini, nanopartikel aragonit

kalsium karbonat dari kulit kerang (CCANPs) digunakan sebagai pengangkut untuk

Cytarabine (Ara-C) sebagai pendekatan unik rawatan kanser. Nanopartikel yang

terhasil daripada gabungan kaedah mekanikal dan kimia adalah berbentuk sfera.

Morfologi dan komposisi CCANPs telah dikenalpasti melalui Mikroskop

Pengimbasan Pelepasan Medan (FE-SEM), Mikrsokop Transmisi Elektron (TEM), X-

ray Penyerak Tenaga (EDX), Transformasi Fourier Spektroskopi Infra Merah (FTIR)

dan Potensi Zeta. Drug anti-leukemia (Ara-C) telah dimuatkan ke dalam CCANPs.

Spektrofotometer dengan gelombang bebas ultra-ungu telah digunakan untuk menilai

jumlah muatan dan profil pelepasan Ara-C. Keputusan menunjukkan drug Ara-C

boleh dimuatkan secara berkesan ke dalam CCANPs dan sebagai tambahan, pelepasan

Ara-C daripada pengangkut nano adalah pantas dan berterusan pada pH 4.8 dan

perlahan pada pH 7.4, di mana ini menunjukkan sifat kecenderungannya terhadap pH.

Pengangkut nanotelah digunakan sebagai pengangkut melawan sel leukemia manusia

HL-60 (kajian in-vitro) dan rawatan kanser menggunakan model xenograf mencit

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(CISD) (di dalam kajian in-vivo). Penilaian in-vitro menunjukkan nilai IC50 selepas

rawatan selama 72 jam dengan Ara-C adalah 5 µg/ml dan Ara-C/CCANPs adalah 2.5

µg/ml. Proses apoptosis telah ditunjukkan melalui reagen Pengiraan Sel (SF),

Sitometer Aliran (FCM), Methylene Biru (MB) dan Mikroskopi Flouresens (FM) di

mana telah jelas pengambilan selular Ara-C/CCANPs ialah melalui endositosis yang

menunjukkan perhubungan respon dos dan masa. Pemerhatian morfologi melalui

SEM menunjukkan kehilangan mikrovili, pengecutan sel, pembengkakan membran

dan pembentukan jasad apoptosis yang mengesahkan kedua-dua Ara-C dan Ara-

C/CCANPs separa dos berjaya menghasilkan apoptosis ke atas HL-60. Secara ringkas,

ARA-C/CCANPs adalah lebih sitotoksik dari Ara-C ke atas sel leukemia HL-60.

Kajian in-vivo menunjukkan pengangkut nano CCANPs berjaya meningkatkan

(P

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ACKNOWLEDGEMENTS

First of all, I thank Allah the Almighty for the great grace. Despite all the difficulties

that faced me during my study, but his great mercy has given me the strength and

perseverance to complete this study. I praise and thank him a lot. I would like to

express my deep gratitude and appreciations towards my respected supervisor Prof.

Dr. Md Zuki Abu Bakar for his wonderful advice, thoughtful guidance and continuous

support. He encouraged me through the difficulties and inspired me to move to a

higher level of the learning experience. I would also like to give my heartfelt thanks

and deepest appreciation and gratitude to my co-supervisors Dr. Intan Shameha Abdul

Razak, Associate Professor Dr. Mohd Hezmee Mohd Noor and Associate Prof. Dr.

Hazilawati Hj Hamzah for the continuous support of my PhD study and related

research, for their patience, motivation, and immense knowledge. I would also like to

express my gratitude to University Putra Malaysia for giving me the opportunity to

complete this research. Without both the financial aids and grants, this thesis may yet

still incomplete. A very special thanks all lecturers, technicians and staffs from

Veterinary Medicine, UPM for their efforts to the successful of this work. I am so

grateful to Iraqi Ministry of Higher Education and scientific research and Basrah

University / Veterinary Medicine / Basrah, for their financial support and gave me the

opportunity to continue my PhD. study. Last but not least, I would like to appreciate

my beloved parents, my siblings, my dearest wife, my beloved kids and my friends for

their unending support throughout this study. Thank you for the love, encouragement

and prayers since the commencement of this study. I would not have made it this far

without them. Thank you all.

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.

The members of the Supervisory Committee were as follows:

Md Zuki Abu Bakar, PhD

Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Chairman)

Intan Shameha Abdul Razak, PhD

Senior Lecturer



(Member)

Mohd Hezmee Mohd Noor, PhD

Associate Professor



(Member)

Hazilawati Hj Hamzah, PhD Associate Professor



(Member)

____________________________

ROBIAH BINTI YUNUS, PhD Professor and Dean

School of Graduate Studies


Date

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xxi

CHAPTER

1 INTRODUCTION 1 1.1 General Background 1 1.2 Problem Statements 2 1.3 Hypothesis 2 1.4 Research Question 2 1.5 Objectives of the Study 3

1.5.1 Main Objective 3 1.5.2 Specific Objectives 3

2 LITERATURE REVIEW 4 2.1 Leukemia 4 2.2 Acute Myeloid Leukemia (AML) 4 2.3 Risk Factors in AML 5 2.4 Epidemiology of AML 5 2.5 Diagnosis of AML 6 2.6 Hematopoiesis 6 2.7 Classification 7

2.7.1 The French-American-British (FAB) Classification of AML 8

2.7.2 World Health Organization (WHO) Classification of

AML 9 2.8 Current Therapies for Leukemia 9

2.8.1 Biological Therapy 10 2.8.2 Targeted Therapy 10

2.8.3 Radiation Therapy 10 2.8.4 Stem Cell Transplant 11 2.8.5 Chemotherapy Drugs 11

2.8.5.1 Cytarabine 11 2.8.5.2 Dose of Cytarabine 13

2.9 Nanoparticles (NPs) 14 2.10 Factors that can Affect on Treatment of Leukaemia 16

2.10.1 Dose of Drug 16

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2.10.2 Size and Shape of Nanoparticles 16 2.10.3 Amount of Drug Loading 17 2.10.4 Drug Release 17 2.10.5 Surface Charge 17

2.11 Calcium Overview Information 17 2.11.1 Calcium Carbonate 18 2.11.2 Type of Calcium Carbonate in Nature 18

2.12 Search Strategy and Selection 19 2.13 Mouse Model 26

2.13.1 Preclinical Mouse Models of Leukemia 26 2.14 Summary 27

3 SYNTHESIS AND CHARACTERISATION OF COCKLE SHELL-DERIVED CaCO3 NANOPARTICLES AND

CONTROLLED RELEASE PROFILE OF CYTARABINE 28 3.1 Introduction 28 3.2 Materials and Methods 29

3.2.1 Preparation of Micron-Size Powder of Cockle Shell-Derived CaCO3 Aragonite (CCAMPs) 29

3.2.2 Preparation of Cockle Shell-Derived CaCO3 Aragonite Nanoparticles (CCANPs) 29 3.2.2.1 Via Mechanical Method 29 3.2.2.2 Via Combination of Chemical and Mechanical

Method 31

3.2.3 Morphology, Crystallinity and Surface Properties of CCANPs 31

3.2.4 Ara-C Loading and Encapsulation Efficiency 32 3.2.4.1 Ara-C Loading (Water Solvent) and

Encapsulation Efficiency 32 3.2.4.1 Ara-C loading (Alcohol Solvent) and the

Encapsulation Efficiency 32 3.2.5 In vitro Controlled Drug Release Study 32

3.3 Results and Discussions 33 3.3.1 Synthesis of CCANPs 33

3.3.2 TEM and FESEM Analysis 33 3.3.3 Determination of λ max of CCANPs 36

3.3.4 Elemental Composition of the Synthesized CCAMPs and CCANPs 37

3.3.5 Powder XRD Analysis 39 3.3.6 FT-IR Spectroscopy 39 3.3.7 Particle Size and Zeta Potential 43

3.3.8 The Ara-C Loading (Water Solvent) and Encapsulation Efficiency 45

3.3.9 The Ara-C Loading (Alcohol Solvent) and Encapsulation Efficiency 45

3.3.10 In vitro Drug Release 48

3.4 Conclusion 49

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4 In vitro EVALUATION OF Cytarabine-LOADED CCANPs FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA 50 4.1 Introduction 50 4.2 Materials and Methods 52

4.2.1 Chemicals and Test Media 52 4.2.2 Cell Culture (HL-60) 52 4.2.3 Storage of HL-60 Cells Seed Stock 52 4.2.4 Cytotoxic Effects of Ara-C, Ara-C/CCANPs and

CCANPs on HL-60 Cells 53 4.2.5 Cellular Uptake Assay 53 4.2.6 Flow Cytometric Analysis of Apoptosis and Necrosis 54 4.2.7 Morphological Changes in HL-60 Cells 54

4.2.8 Statistical Analysis 54 4.3 Results and Discussion 54

4.3.1 Cell Activity (Cell Counter Reagent (SF) Assay) 54 4.3.2 Evidence of Apoptosis by Flow Cytometry (FCM) 57 4.3.3 Evidence of Apoptosis by Fluorescence Microscopy 61 4.3.4 Evidence of Apoptosis by SEM 64 4.3.5 Anticancer Potential 65

4.4 Conclusions 65

5 In vivo RELEASE OF CYTARABINE-LOADED COCKLE SHELL-DERIVED CALCIUM CARBONATE ARAGONITE

NANOPARTICLES (ARA-C/CCANPs) 66

5.1 Introduction 66 5.2 Materials and Methods 67

5.2.1 Calibration Curve of Ara-C 67 5.2.2 In Vitro Release of Ara-C 68 5.2.3 Stability Studies of Ara-C/CCANPs 68 5.2.4 In vivo Release of Ara-C 69

5.2.4.1 Experimental Animals 69 5.2.4.2 Pharmacokinetic Study 69 5.2.4.3 HPLC Linearity 69 5.2.4.4 Preparation of Stock Solutions and Quality

Control of Samples 70 5.3 Results and Discussion 70

5.3.1 In vitro Release of Drug 70 5.3.2 Stability Studies of Ara-C/CCANPs 70 5.3.3 Linearity and Quantification and Limits of Detection 71 5.3.4 Pharmacokinetic Study 73

5.4 Conclusion 76

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6 EFFECTS OF CYTARABINE-LOADED CALCIUM CARBONATE ARAGONITE NANOPARTICLE ON ACUTE

MYELOID LEUKEMIA In vivo: A NEW APPROACH TOWARDS

INCREASING THE SPECIFICITY AND REDUCING THE

TOXICITY OF ANTI-LEUKEMIA DRUG 77 6.1 Introduction 77 6.2 Materials and Methods 79

6.2.1 HL-60 Cell Line 79 6.2.2 Animal Xenograft Model and Experimental Design 79 6.2.3 Histopathological Examination 82 6.2.4 Anesthesia of Mice 82 6.2.5 Blood Collection 82

6.2.6 Wright Staining of Blood Smears 83 6.2.7 Haemato-Toxicity Evaluation 83 6.2.8 Serum Biochemistry Evaluation 83 6.2.9 Bone Marrow Smear 83

6.3 Results and Discussion 84 6.3.1 Establishment and Characterization of

Xenotransplantation 84 6.3.2 Body Weight of Mice 84 6.3.3 Weight of Organs 85 6.3.4 Histopathological Examination 86

6.4 Hematological Parameters 103 6.4.1 Serum Biochemistry 109

6.4.2 Bone Marrow Smear 112 6.5 Conclusion 115

7 GENERAL DISCUSSION 116 7.1 Conclusion 118 7.2 Recommendations for Future Research 118

REFERENCES 119 APPENDICES 148 BIODATA OF STUDENT 158

LIST OF PUBLICATIONS 159

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LIST OF TABLES

Table Page

2.1 The French-American-British (FAB) classification of AML 8

2.2 The classification of AML based on the WHO 2008 criteria 9

2.3 Data extracted from the previous studies on drug nanocarriers 21

3.1 Elemental compositions of CCANPs. 38

3.2 Elemental compositions of CCAMPs. 38

3.3 Drug loading and encapsulation efficiency of Ara-C into CCANPs 45

3.4 Drug loading and encapsulation efficiency of Ara-C into CCANPs 46

4.1 Cell viability percentage of Ara-C and Ara-C/CCANPs on HL-60 cells

determined by FS assay at 24, 48 and 72 h 57

5.1 Stability evaluation of Ara-C-CCANPs at 4°C, room temperature (25°C)

and 40°C 71

5.2 Pharmacokinetic profile of Ara-C in BALB/c mice plasma 72

5.3 Data shows drug release study for Ara-C in plasma mice. Amount of

drug released was determined from the calibration curve. * represent

significant difference at p˂0.05 73

6.1 Total WBC, differential white blood cell, RBC and PCV in SCID mice

for all experimental groups. The results presented are Mean ± SE (n=3) 111

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LIST OF FIGURES

Figure Page

2.1 A normal hematopoietic process. This figure depicts the normal

differentiation of HSCs into the respective blood components; this is an

essential leukemic process 7

2.2 Cytarabine(4-amino-1-ß-D-arabinofuranosyl-2 (1H)-pyrimidinone).

Cytosine normally combines with Ribose/Deoxyribose sugar, has a

molecular weight of 243.22, and a molecular formula of C9H13N3O5 13

2.3 : Preferred reporting items for systematic reviews and meta-analyses flow

diagram: screening procedure of selected articles 20

3.1 Diagrams show the jar with strips of aluminum bars and cross-section

show barrier and glass balls with CCANPs 30

3.2 Diagram shows the Roll Mill with jar on the cylindrical (horizontal

position) 30

3.3 TEM micrograph of CCAMPs magnification 100 nm (A) and FESEM

micrograph of CCAMPs were sized at a rate of 1mm and magnification

X50000 (B) 34

3.4 FESEM micrograph of (A) CCANPs prepared via mechanical method

and (B) Ara-C/CCANPs were sized at a rate of 30nm and magnification

X2000000 34

3.5 TEM micrograph of CCANPs prepared via a mechanical method and the

particles were sized at a rate of 40 nm (A). Ara-C/CCANPs (B),

magnification X50000 35

3.6 FESEM micrographs of (A) CCANPs prepared via a combination

method and (B) Ara-C/CCANPs were sized at a rate of 25nm and

magnification X100000 35

3.7 TEM micrographs of (A) CCANPs prepared via a combination method

and (B) Ara-C/CCANPs were sized at a rate of 35nm and magnification

X50000 36

3.8 The wavelength of Ara-C in the UV spectrum 37

3.9 EDX spectral of CCANPs (A) and CCAMPs (B) 38

3.10 XRD pattern for (A) Ara-C, (B) CCAMPs, (C) Ara-C/CCANPs and (D)

CCANPs 39

3.11 FTIR spectra of Ara-C/CCANP (A), CCANPs (B) and Ara-C pure (C) 41

3.12 FTIR spectra of CCMPs 42

3.13 Particle size distributions (PSD) for CCANPs (A); Zeta potential of

CCANPs before loaded (B) and after loaded (C) with Ara-C 44

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3.14 FTIR spectra of CCANPs (40 mg), with different concentration of Ara-

C (10, 20, 40 mg) 47

3.15 Drug release profile for Ara-C, and Ara-C/CCANPs. Each point is the

average of three replications and the vertical bars represent standard

deviations 48

4.1 Relative cell counts of HL-60 cells incubated at various concentrations

for 24, 48 and 72hr. Values represent the mean ± standard error of the

mean (n = 3); denotes statistically significant differences from control

(*p < 0.05) 56

4.2 Results of Flow cytometry showing a different distribution of cell

cytopathology in HL-60 cells (control) and treated with Ara-C alone and

½ Ara-C/CCANPs at 24, 48, and 72 h. viable cells are red; early

apoptosis is green; late apoptosis is blue 59

4.3 Quantitative estimation (%) of cell cytopathology in HL-60 cells

untreated and treated with Ara-C (5µM/mL) and Ara-C/CCANPs

(2.5µM/mL) showing percentages of viable cells (VC), early apoptosis

(EA), late apoptosis (LA) and necrosis (N) at 24, 48, and 72 h. Bars

with a different number of asterisks are significantly different at p <

0.05 60

4.4 Fluorescent micrographs of acridine orange/propidium iodide double-

stained HL-60 cells treated with (A) pure Ara-C, (B) ½ Ara-C/CCANPs

and (C) Untreated cells without any morphological changes, (A and B)

Cells treated for 72 hours show more orange-colored staining, indicating

early apoptosis (yellow arrows); X100. 63

4.5 Scanning electron micrograph of (A) untreated cell, (B) Ara-C 5µM/ml,

(C) Ara-C 2.5µM/ml loaded into CCANPs 5µg/ml and (D) Ara-C

5µg/ml loaded into CCANPs 10µg/ml treated groups. Treatment groups

show the presence of blebbing signaling apoptosis 64

5.1 Cytarabine structure and formula C9H13N3O5 67

5.2 Calibration curve of Ara-C (Spectrophotometer correlation = 0.995) 68

5.3 Stability study of Ara-C-CCANPs at 4 °C, 25 °C and 40 °C 71

5.4 Calibration curve of Ara-C by HPLC (Correlation = 0.998) 72

5.5 Data shows drug release study for Ara-C in plasma mice. The pure Ara-

C levele in the plasma was higher (p

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6.1 Diagram showing the method used in the induction of leukemia in SCID

mice 80

6.2 Summary of the experimental design 81

6.3 Graft shows animals in the experimental groups loss their weight before

treatment, and then regain their weight once the treatment begin except

for the negative control group. The normal control group continued to

gain weight. The values are mean ± SD of four measurements (n = 4) for

five groups analyzed using test one way ANOVA 85

6.4 Weight of organs of all groups experience at the end of the experimental

period. Data are normalized to Negative control group and expressed as

mean ± SE of (n=4). *p < 0.05 compared with normal control and all

treatments 86

6.5 Photomicrograph of the liver from NOD/SCID mice of normal control

group showing normal structure. Central vein. H&E; X200 87

6.6 Photomicrograph of the liver from NOD/SCID mice induced AML

without treatment in negative control group at 53 days shows the area of

metastatic tumor cells (MTC) (1), necrosis (black arrows) and liver cell

degeneration (yellow arrows). H&E; X200 87


treated with Ara-C at 40 days of treatment shows sign of liver toxicity.

Congestion (1) and hemorrhage (2). H&E; X200 88


treated with Ara-C/CCANPs (50mg/kg Ara-C-loaded CCANPs

100mg/kg) at 40 days of treatment shows moderate changes with no

evidence of MTC. Slight heamorrage (1); and necrosis (2). H&E, X200 88


treated with ½ dose of Ara-C/CCANPs at 40 days of treatment shows

mild changes with no evidence of MTC. Normal sinusoidal (1);

vacuolation (2). H&E, X200. 89

6.10 Photomicrograph of the lung from NOD/SCID mice of normal control

group showing normal alveoli and bronchioles (1). H&E, X100 90

6.11 Photomicrograph of the lung from NOD/SCID mice induced AML


MTC (1), congestion (2) and alveolar wall degeneration (3). H&E, X100

90


treated with Ara-C at 40 days of treatment shows the alveolar wall

degeneration (1) and congestion (2) with no evidence of MTC. H&E,

X100 91

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100mg/kg) at 40 days of treatment shows the evidence of congestion

with no area of MTC. H&E, X200 91


treated with ½ Ara-C/CCANPs (25mg/kg Ara-C-loaded CCANPs

50mg/kg) at 40 days of treatment shows the evidence of congestion with

no area of MTC. H&E, X200 92

6.15 Photomicrograph of the kidney from NOD/SCID mice of normal control

group shows normal structure of kidney. H&E, X400 93

6.16 Photomicrograph of the Kidney from NOD/SCID mice induced AML

without treatment in negative control group at 53 days shows area of

MTC (1), tubule necrosis (2) and hemorrhage (3). H&E, X400 93


treated with Ara-C alone at 40 days of treatment shows epithelial cells

degeneration (1), tubule vaculation (2) and hemorrhage (3). H&E, X400

94



100mg/kg) at 40 days of treatment shows tubule necrosis (1) and

hemorrhage (2). H&E, X200. 94


treated with ½ Ara-C/CCANPs at 40 days of treatment show tubule

necrosis (1) and hemorrhage (2). H&E, X100 95

6.20 Photomicrograph of the heart tissue from NOD/SCID mice of normal

control group showing normal heart wall structure. H&E X200 96

6.21 Photomicrograph of the heart from NOD/SCID mice induced AML


MTCs (1), vacuolation (2) and myocardial degeneration (3). H&E, X200

97

6.22 Photomicrograph of the heart tissue from NOD/SCID mice induced

AML treated with Ara-C at 40 days of treatment shows congestion (1)

and myocardial degeneration (2) . H &E X200 97


AML treated with Ara-C/CCANPs (50mg/kg Ara-C-loaded CCANPs

100mg/kg) at 40 days of treatment shows hemorrhage (1) and vaculation

(2). H&E; X200 98


AML treated with ½ dose Ara-C/CCANPs at 40 days of treatment shows

hemorrhage. H&E; X200 98

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6.25 Photomicrograph of the spleen from NOD/SCID mice of normal control

group showing the normal structure. White pulp (1), red pulp (2) and

central arteriole (3). H&E; X100 99

6.26 Photomicrograph of the spleen from NOD/SCID mice induced AML


MTC (yellow arrows), borderline lesion was diagnosed as loss of cells

in the marginal zone (1). H&E; X400 100


treated with Ara-C at 40 days of treatment shows extra-medullary

erythroid haemopoiesis. Note the aggregation of erythroid cells in the

red pulp. H&E; X400 100



100mg/kg) at 40 days of treatment shows loss of cells in the marginal

zone (black arrows). H&E; X200 101


treated with ½ dose Ara-C/CCANPs at 40 days of treatment shows loss

of cells in the marginal zone. H&E; X200 101

6.30 Photomicrograph of the blood smear from NOD/SCID mice induced

AML without treatment in negative control group at 14 days after cancer

induction shows high number of leukocyte. Wright staining, X400 104


AML without treatment in negative control group at 53 days shows

increase number of leukocyte. Wright staining, X400 104

6.32 Graph shows the total number of RBC of mice at day 53. Data are

normalized to negative control group and expressed as mean ± SE of

(n=4). *p < 0.05 compared with negative control group 105

6.33 Graph shows the total number of WBC of mice at day 53. Data are

normalized to Negative control group and expressed as mean ± SE of

(n=4). *p < 0.05 compared with normal control and all treatments 106


AML treated with ½ Ara-C/CCANPs at 53 days shows normal range of

WBC. Wright staining, X400 106


AML treated with Ara-C/CCANPs (50mg/kg Ara-C-loaded CCANPs

100mg/kg) at 53 days shows normal range of WBC. Wright staining,

X400 107

6.36 Photomicrograph of the blood smear from NOD/SCID mice of normal

control group showing normal range of WBC. Wright staining, X400 107


AML treated with Ara-C at 53 days shows normal range of WBC.

Wright staining, X400 108

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6.38 Graph shows of packed cell volume (PCV) of mice at day 53. Data are


(n=4). *p < 0.05 compared with negative control group 108

6.39 Differential WBC across treatment groups at day 53. *p < 0.05 compared

with another groups 109

6.40 Graph shows the amount of urea in mice at day 53. Data are normalized

to Negative control group and expressed as mean ± SE of independent

trials. *p < 0.05 compared with normal control and all treatments 110

6.41 Graph shows the total amount of Bilirubin in mice at day 53. Data are


independent trials. *p < 0.05 compared with normal control and all

treatments 110

6.42 Photomicrograph of the bone marrow of NOD/SCID mice (normal

control group) Wright’s–Giemsa, X200 113

6.43 Photomicrograph of the bone marrow of NOD/SCID mice induced AML

treated with Ara-C at 40 days of treatment shows polymorphonuclear

cell (1), fat tissue (yellow arrows), erythroblast (2) and lymphoblast (3).

Wright’s–Giemsa; X200 113

6.44 Photomicrograph of the bone marrow NOD/SCID mice induced AML


100mg/kg) at 40 days of treatment shows fat tissue (yellow arrows),

erythroblast (1), and lymphoblast (2). Wright’s–Giemsa; X200 114

6.45 Photomicrograph of the bone marrow NOD/SCID mice induced AML

treated with ½ Ara-C/CCANPs at 40 days of treatment shows

polymorphonuclear cell (1), fat tissue (yellow arrows), erythroblast (2)

and lymphoblast (3). Wright’s–Giemsa; X200 114

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LIST OF ABBREVIATIONS

AML Acute myeloid leukemia

ALL Acute lymphoid leukemia

CML Chronic Myeloid Leukemia

CLL Chronic Lymphoid Leukemia

Ara-C Cytarabine

EDX Equipped dispersive x-ray

DL Drug loaded

LE Loading efficiency

FESEM Field emission scanning electron microscopy

FTIR Fourier transformed infrared

CCANPs Calcium Carbonate Aragonite nanoparticles

Ara-C/CCANPs Cytarabine-loaded Calcium Carbonate nanoparticles

NP Nanoparticle

PBS Phosphate Buffer Saline

SEM Scanning electron microscopy

TEM Transmission electron microscopy

XRD X-ray diffraction

Nm Nano meter

Mg/m2 Milligram per square meter

mL Milliliter

µ Micro meter

Kg Kilogram

nM Nano Molar

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µg/mL Microgram per Milliliter

h Hour (s)

IC50 Lethal concentration, 50%

SCID mice Severe combined immunodeficient mice

CO2 Carbon dioxide

AO Acridine Orange

ATCC American Type Culture Collection

FBS Fetal bovine serum

Min Minute

Rpm Revolution per minute

PI Propidium iodide

UV Ultraviolet

% Percentage

DNA Deoxyribonucleic acid

HD High dose

LD Low dosage

BM Bone marrow

PSD Particle size distributions

MTD Maximally tolerated dose

ID Intermediate dose

MTCs Metastatic tumour cells

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CHAPTER 1

1 INTRODUCTION

1.1 General Background

Cancers develop from an uncontrolled growth of body cells and can develop from any

part of the body. Any tissue from any part of the body can become cancerous provided

the necessary factors needed for its propagation are in place (Folkman, 1996).

Leukemia is a cancerous condition which primarily affects the blood and bone

marrow. In the case of acute myeloid leukemia (AML), it develops from the bone

marrow and in most cases, spread to the blood system. AML can also migrate from

the bone marrow to the spleen, the central nervous system, the liver, the testicles, and

the lymph nodes (Valent et al., 2007). The global rate of leukemia cases in 2012 was

put at 4,000,000, resulting in 3,000,000 deaths. A large number of people in the USA

(around 24,500, made up of 14,300 males and 10,200 females) were predicted to die

from leukemia-related conditions in 2017 (Siegel et al., 2017). AML is the commonest

form of adult leukemia, which despite the intimidating statistics regarding its

associated mortality and morbidity, the treatment options is still unsuccessful in its

management. Chemotherapy, which aims at killing the dividing cells over time to

restore the normal blood count of normal cells, has been the only option for AML

management; but, chemotherapy is a highly toxic procedure which lacks specificity.

Nano-carriers are a new method of improving the specificity of chemotherapeutic

agents and reducing their toxicity level. Based on this, cockle shell (Anadara

granosa)-derived calcium carbonate aragonite nanoparticles (CCANPs) was produced

and used as a drug carrier in this study. The size of nanoparticles (NPs) was within the

transitional zone between the corresponding bulk materials and the individual atoms

or molecules. This helps to alter the material’s physicochemical properties and present

a chance to increase the uptake capability and interaction with biological tissues. In

the living cells, the combination of these events can have an adverse impact

biologically, which otherwise, would not have been possible with the same material

in a larger form (Moore, 2006). Calcium and its derivatives are the most vital

components of teeth and bone, in fact, the osseous tissues of bone are primarily

composed of inorganic calcium-derived composite materials (Bandyopadhyay-Ghosh,

2008). Calcium carbonate (CaCO3) is the commonest calcium derivative with the

longest history of applications in various fields, such as in the plastics, paper, paint,

food, inks, and pharmaceutical industries (Biradar et al., 2011). In the modern times,

CaCO3 has attracted medical attention owing to its high applicability. It is a cost-

effective, safe, biocompatible, resorptive, accessible, and osteoconductive material

(Biradar et al., 2011). Owing to its pH sensitivity and relatively slow degradation, it

can be used as an agent for the controlled release of active substances such as drugs to

maintain their tolerable serum concentration and for targeted delivery over time (Qian

et al., 2011). This study focused on the synthesis, characterization, and application of

CCANPs as an agent for the in vivo and in vitro controlled release of Ara-C to targeted

cancer tissues.

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1.2 Problem Statements

Acute leukemia is an aggressive form of cancer that requires immediate treatment.

AML multimodal chemotherapy is used to re-establish and normalize blood and bone

marrow cell numbers and morphology. Ara-C is a traditional chemotherapeutic agent

for the management of all types of leukemia (Gökbuget et al., 2011). It is similar to

most cancer chemotherapies which target the S-phase of cell division (healthy and

cancer cells). The strategy requires a prolonged period of cell exposure to highly toxic

concentrations of the agents for cancer treatment (Gökbuget et al., 2011; Hamada et

al., 2002). However, the activity of Ara-C is decreased by its rapid deamination to the

biologically-inactive metabolite, uracil (Hamada et al., 2002). This rapid deamination

led to a search for effective formulations of Ara-C that cannot be deaminated, but still,

exhibit better pharmacokinetic parameters and protection for Ara-C. The chemical

therapies for AML are often limited in their use by high systemic toxicity and low

specificity. Drug delivery through carrier systems is presumed to avoid their side

effects through a controlled biodistribution. These carriers can contribute towards the

control of leukemia metastasis. A new natural approach at nano-scale needs to be

developed which ensures an efficient and enhanced drug delivery for AML treatment.

1.3 Hypothesis

i. CCANPs is a biocompatible and non-toxic to normal cells in normal pH.

ii. CCANPs can be used as nano-carrier in the management of AML.

iii. CCANPs reduce effective dose of Ara-C into half.

iv. CCANPs loaded Ara-C has therapeutic effects on reduced metastasis HL-60

human cells to other organs.

1.4 Research Question

i. What is the in vitro drug release profile and biocompatibility of CCANPs

loaded Ara-C?

ii. How safe is CCANPs loaded Ara-C on the biological system in vivo?

iii. How effective CCANPs loaded Ara-C in the treatment Acute Myeloid

leukaemia?

iv. How CCANPs loaded Ara-C can reduce metastasis AML in other body

organs?

v. How CCANPs loaded Ara-C can reduce the dose of Ara-C?

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1.5 Objectives of the Study

1.5.1 Main Objective

This study was conducted with the aim of investigating the effectiveness of CCANPs

loaded Ara-C in the treatment of AML.

1.5.2 Specific Objectives

i. To synthesise and characterize CCANPs, and evaluate the in-vitro release profile of CCANPs loaded Ara-C.

ii. To determine the CCANPs loaded Ara-C in the treatment HL-60 human cells in vitro.

iii. To determine the pharmacokinetic of CCANPs loaded Ara-C in vivo. iv. To evaluate the effectiveness of CCANPs loaded Ara-C in the treatment of

AML in SCID mice-induced AML in vivo.

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